Youn-Sun Lee1, Heon-Kyo Choi1, Jae-Myung Yoo1, Kyong-Mi Choi1,
Yong-Moon Lee1, Seikwan Oh2, Tack-Joong Kim1, Yeo-Pyo Yun1, Jin-Tae Hong1, Nozomu Okino3, Makoto Ito3& Hwan-Soo Yoo1
1College of Pharmacy and CBITRC, Chungbuk National University, Cheongju 361-763, Korea
2Department of Neuroscience, College of Medicine, Ewha Womans University, Seoul 158-710, Korea
3Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan
Correspondence and requests for materials should be addressed to H. S. Yoo (yoohs@chungbuk.ac.kr)
Accepted 6 November 2007
Abstract
Ceramide is involved in cell death as a lipid media- tor of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine (C17 ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acid- free bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and mea- sured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separa- tion of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSA- ceramide complex leads to the increased deacyla- tion of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be appli-
cable to diverse biological samples.
Keywords: Ceramide, HPLC, Cell death, ICR mice, LLC- PK1 cells
Ceramide is involved in the regulation of cell death1 and acts as a lipid mediator of cellular stress respons- es2. The ceramide level in cells is up-regulated by various types of stress conditions including ionizing radiation3, serum deprivation4,5and anti-cancer drugs6. There are two potential pathways for intracellular ceramide formation: de novo biosynthesis via the condensation of serine and palmitoyl-CoA, and the breakdown of sphingomyelin via sphingomyelinase.
Many analytical methods for ceramide quantifi- cation in biological samples have been published. In the radiolabelled method, ceramide is phosphorylated into [32P]ceramide 1-phosphate by E. coli diacylgly- cerol (DAG) kinase and [γ-32P]ATP, and the amount of ceramide is calculated by measuring the radioac- tivity on a thin-layer chromatography (TLC) plate and compared to a standard curve7. Although this is a sensitive method that is used by most researchers, its disadvantages include the use of radioisotopes and the lack of an internal standard.
An alternative method involves the use of mass spectrometry for the quantitative analysis of ceramid- es following extraction of all lipids8,9. Although each class of ceramide can be quantified with high sensi- tivity, this method requires the use of expensive equipment that is not available in most laboratories.
Another method uses HPLC analysis of ceramide following lipid extraction and ceramide deacylation with CDase10. Although detection with this method is sensitive and the procedure is simple, the absence of purification step and an incomplete solubility of cera- mide in deacylation reaction, and different extraction efficiencies due to no internal standard may cause variation for the quantification of ceramide.
Here we present an improved method with high specificity and good reproducibility for determining the amount of ceramide and dihydroceramide in samples, using TLC separation, enzymatic deacyla- tion and HPLC analysis. It is applicable to a variety of biological samples, the use of an internal standard makes it reproducible and TLC separation may pro-
N-oleoyl-D-erythro-sphingosine-based Analysis of Ceramide by High Performance Liquid Chromatography and Its
Application to Determination in Diverse Biological Samples
vide HPLC chromatogram with clean background without any interference.
TLC Separation of Ceramide from Lipid Extract and HPLC Analysis
Ceramide was specifically deacylated by CDase, and sphingosine was released and analyzed by HPLC following OPA derivatization (Figure 1A). Peaks of synthetic C17 ceramide and naturally occurring C18
ceramide on the HPLC chromatogram were shown at the retention times of 9.0 and 11.3 min, respectively (Figure 1B). The dihydroceramide peak on the HPLC chromatogram had a retention time of 15.2 min alth- ough it was not added to CDase reaction, indicating that the natural ceramide standard purified from por- cine brain may contain the trace amounts of dihy- droceramide. C17ceramide as an internal standard for the determination of endogenous ceramide concen- tration was added to the biological samples and lipids were extracted. Ceramide was separated from other sphingolipids by TLC. For the determination of cera- mide contents in the samples, a TLC plate loaded with the C17 ceramide standard in the end lanes and
samples in the inside lanes was developed with a mixture of diisopropylether/methanol/29% NH4OH (40 : 10 : 1, v/v/v). The ceramide standard lanes were cut from the TLC plate and the ceramide standard band was visualized with sulfuric acid reagent.
Ceramide and dihydroceramide appeared as the same band as the C17ceramide standard on the TLC plate with an Rfvalue of 0.54 indicating that both ceramide and dihydroceramide could be simultaneously quanti- fied. Possible contamination of the lipid extract with sphingosine and sphinganine was excluded by the TLC separation. The Rfvalues of sphingosine, sphin- ganine and phytosphingosine are 0.09, 0.06 and 0.04, respectively (Figure 2). Therefore, sphingoid bases did not interfere with the ceramide analysis. Separa- tion of ceramide from lipid extract by TLC provides HPLC chromatogram with clean background (Figure 3). The chromatogram showed many non-specific peaks without ceramide peaks when the lipid extract from human plasma was analyzed by HPLC follow- ing ceramide deacylation without TLC separation (Figure 3A). Thus, ceramide purification from the lipid extract of human plasma by TLC led to the good separation of ceramides on the HPLC chromatogram (Figure 3B). Spiking the biological samples with C17
ceramide as an internal standard overcame the varia- tion which could be caused by the multistep proce- dure including lipid extraction, TLC separation, en- zymatic deacylation, and HPLC quantification.
Optimization of Enzymatic Deacylation and Reproducibility of Ceramide Quantification
CDase reaction was increased in the BSA con- centration-dependent manner ranging from 0 to 15%
in the reaction buffer (Figure 4). Ceramide deacyla-
NH OH
O OH
NH2
OH OH
CDase 37°C, 6 h
HPLC analysis Sphingosine
OH O
Ceramide
Fatty acid
(A)
Figure 1. The procedure for ceramide quantification. (A) Following TLC separation ceramides were deacylated by neutral ceramidase (CDase), and the released sphingosine was derivatized with OPA fluorescence dye and analyzed by HPLC. (B) The retention times for C17 and C18 ceramide standards were 9.0 and 11.3 min, respectively, on HPLC chromatogram. Dihydroceramide was detected at 15.2 min.
Figure 2. TLC chromatogram of sphingolipids. Sphingoli- pids on the TLC chromatogram include C17 and C18cera- mides, dihydroceramide, sphingosine, sphinganine and phy- tosphingosine. Sphingolipid standards were spotted on the TLC plate, and developed in diisopropylether/methanol/29%
NH4OH (40 : 10 : 1, v/v/v) and visualized with 10% sulfuric acid.
1.0E++06
8.0E++05
6.0E++05
4.0E++05
2.0E++05
0.0E++00
C17ceramide
C18ceramide
Dihydroceramide
5.00 10.00 15.00 20.00
Retention time (min)
Fluorescence intensity
(B)
Dihydroceramide C18ceramide
C17ceramide
1 2 3 4 5 6
Sphingosine Sphingansine Phytosphingosine
tion by CDase at 15% BSA was maximal. Deacyla- tion of C17 and C18 ceramides was dependent on the incubation time until 6 hrs (Figure 5A). Thus, the incu- bation time for ceramide deacylation with 150 µ units of CDase was set at 6 hrs. Deacylation of C17and C18
ceramides was concentration-dependent at the range of 5-1,000 pmol with linearity of 0.9982 and 0.9968, respectively, between ceramide concentration and peak area (Figure 5B). Thus, the quantitative analysis of ceramide in human plasma was performed by comparison of the peak area on the chromatogram, and calculated based on 200 pmol of added C17cera- mide as the internal standard. Although the ceramide band on the TLC plate was visually detected at the level of nmol level after spraying with sulfuric acid and heating the plate, the analytical method using
HPLC equipped with a fluorescence detector lowered the detection limit to the 1-2 pmol range, indicating that the method used in this study was very sensitive.
The sensitivity was achieved by the OPA-fluores- cence derivatization of either sphingosine or sphin- ganine. Ceramide recovery was measured by addition of the ceramide standard to human plasma. The level of ceramide showed a linear relationship between the added and the measured amounts, and the recoveries Figure 3. HPLC profiles of ceramides from human plasma.
Blood samples were obtained from healthy volunteers and plasma was prepared by centrifugation at 3,000×g for 10 min. Lipids were extracted from 50 µL of plasma. (A) Cera- mides were deacylated by CDase and analyzed by HPLC.
(B) Ceramides were separated from lipid extracts by TLC, deacylated and quantified.
0 7.5 15 30
*
**
BSA (%)
0 7.5 15 30
*
**
BSA (%) 0
50 100 150 200
0 50 100 150 200 C17 ceramide peak area(×105)C18 ceramide peak area(×105)
(A)
(B)
Figure 4. Effect of BSA on ceramide deacylation. (A) C17
and (B) C18ceramide standards were hydrolyzed by CDase in a reaction buffer at concentrations of 0, 7.5, 15 and 30%
fatty-acid-free BSA for 6 hrs. Sphingosines released from ceramides were analyzed by HPLC. Values are expressed as the mean±S.D. (n==3-5). Difference with *P⁄0.05 and **P
⁄0.01 were defined as statistically significant between un- treated and treated samples.
1.0E++06
8.0E++05
6.0E++05
4.0E++05
2.0E++05
0.0E++00
Fluorescence intensityFluorescence intensity
1.0E++06
8.0E++05
6.0E++05
4.0E++05
2.0E++05
0.0E++00
C18ceramide
C17ceramide
Dihydroceramide
5.00 10.00 15.00 20.00
Retention time (min)
5.00 10.00 15.00 20.00
Retention time (min)
(A)
(B)
were in the range of 86 to 93% (Table 1).
Quantification of Ceramide and
Dihydroceramide in Human Plasma and Mouse Tissues
Ceramide concentrations were determined in 10- 200 µL of human plasma with the addition of 200 pmol C17ceramide. Although ceramide quantification was possible with 10 µL human plasma, the optimum range for ceramide determination appeared to be 50- 150 µL. The concentration of endogenous ceramide in healthy human plasma is approximately 5 µM (Table 2). The levels of total ceramide in ICR mouse tissues varied from 0.8 to 4.0 nmol per mg protein (Table 2). Brain and intestine had the highest levels
of ceramide and liver, spleen, muscle, and heart had the lowest levels. Since ceramide is known as a sig- naling molecule for cell death, the ceramide levels in animal tissues may be related to the organ’s sen- sitivity to toxicity.
Close Relationship between the Elevated Level of Ceramide Content and Cell Death in LLC-PK1 Cells
There was a relationship between released LDH activity and ceramide content in LLC-PK1 cells.
Released LDH activity, an indicator of cytotoxicity, was increased in a concentration-dependent manner by etoposide at 10, 20 and 50 µM in the absence of FBS (Figure 6A). Under the same conditions, total
0.5 1 2 4 8 16 24 36 48
0 5 10 15 20
Deacylation time (h) C17 ceramide
C18 ceramide
C17 ceramide C18 ceramide Ceramide peak area(×105)Ceramide peak area(×105)
0 200 400 600 800 1000 1200
0 25 50 75 100 125 150
Y=0.134X, R2=0.9982 Y=0.123X, R2=0.9968
Ceramide concentration (pmol)
(A)
(B)
Figure 5. Optimal conditions for ceramide deacylation by CDase. (A) The time-dependent release of sphingosine and sphinganine from ceramide standards. C17and C18ceramides (200 pmol each) were added to the reaction buffer and incu- bated at 37°C for varying periods of time. The released sphin- gosine and sphinganine were analyzed by HPLC. (B) Linea- rity between ceramide concentration and fluorescence peak area at the concentrations of 5-1,000 pmol. The CDase dea- cylation of C17 and C18 ceramides was continued for 6 hrs.
Values are expressed as the mean±S.D. (n==3-5).
Table 1. Recovery of ceramide in human plasma.
Added ceramide Ceramide
solution (µL)a Measured Expressed Recovery
(µM)b (µM) (%)
0 5.01
3 5.94 6.81 87
6 8.09 8.61 93
9 9.56 10.41 91
15 12.61 14.01 90
30 19.83 23.01 86
aMethanolic solution with 30 µM natural ceramide
bValues are the means of triplicate measurements
Table 2. Levels of ceramide and dihydroceramide in cultur- ed cells, plasma and tissues.
Sources Ceramidesa Dihydroceramides Cell type
HeLa 3.54±0.18 0.20±0.02
VSMC 3.08±0.15 0.13±0.02
LLC-PK1 1.94±0.08 0.12±0.01
Plasma
Human 5.01±0.25 0.21±0.11
ICR mice 4.68±0.21 0.22±0.02
Tissues (ICR mice)
Brain 4.18±0.17 0.50±0.26
Intestine 2.20±0.10 0.19±0.07
Testis 2.07±0.03 0.32±0.05
Lung 1.53±0.06 NDb
Kidney 1.35±0.06 0.07±0.02
Eye 1.21±0.05 ND
Spleen 0.82±0.05 ND
Heart 0.75±0.02 ND
Liver 0.72±0.03 ND
Muscle 0.61±0.01 ND
aValues are in nmol/mg protein for cultured cells and tissues and µM for plasma and are means±SDs; n==3
bND, not detected
ceramide increased in a concentration-dependent manner up to 50 µM (Figure 6B). In the absence of FBS, the ceramide concentration in LLC-PK1 cells increased about 3.8-fold, from 1.9 nmol to 7.2 nmol
per mg protein, with 50 µM etoposide treatment.
However, ISP-1, an inhibitor of serine palmitoyl- transferase in the de novo sphingolipid biosynthesis pathway, partially protected the cells from the serum- starvation-induced cell death (Figure 7A). This result suggests that cell death under serum deprivation may occur via sphingolipid metabolism. Prolonged serum deprivation elevated the intracellular ceramide con- centration by 2.2-fold, from 2.0 nmol in the presence of FBS to 4.4 nmol in the absence of FBS (Figure 7B). ISP-1 partially reduced the elevated ceramide level by 63%, indicating that the activation of de novo ceramide biosynthesis may contribute to serum- deprivation-induced cell death in LLC-PK1 cells.
Discussion
Ceramide is involved in cell death1 as a lipid me- diator of stress responses2and has been implicated in human diseases such as Alzheimer’s disease11,12 and cancer6,13. Therefore, several methods have been de- veloped for ceramide quantification, including the ra- dioactive DAG kinase assay7,14,15 and mass spectro- metry method8,9. However, these methods are depen- dent on either radioisotopes or expensive instruments.
Mass spectrometry analysis provides quantification of molecular species of ceramides with high sen- sitivity. However, the sensitivity can be dependent on the mode of ionization and may vary with the mole- cular species. A recent method for ceramide quanti- fication utilizes HPLC, following CDase deacylation and naphthalene-2, 3-dicarboxyaldehyde fluorescence derivatization10. Although this method appears to be simple and sensitive, variations due to different ex- traction efficiencies remain. Another method utilizes microwave-assisted deacylation of ceramide, gluco- sylceramide, and ceramide trihexoside to measure lysosphinogolipids by HPLC16,17. However, the use of sphinganine as an internal standard in the microwave deacylation method is not appropriate for ceramide quantification because small amounts of sphinganine exist in biological samples.
Our procedure includes the TLC separation of cera- mide, hydrolysis of ceramide to sphingosine using Pseudomonas CDase18, OPA fluorescence derivatiza- tion, and HPLC analysis (Figure 1B, 3B). In addition, spiking with C17 ceramide as an internal standard compensates for variations in the extraction of endo- genous ceramide, the degree of enzymatic hydrolysis, and differences in sensitivity. With this method, the analytical detection limit for ceramide is shown to be 1-2 pmol and the optimal amount of sample is either cell lysate equivalent to 100 µg total protein or 50-
0 10 20 50 100
Etoposide (µM)
*
**
*** ***
Released LDH activity(% of total)
0 10 20 50 100
*
**
*** ***
Ceramide(nmol/mg protein)
0 10 20 30 40 50 60 70
0.0 2.5 5.0 7.5 10.0
Etoposide (µM)
(A)
(B)
Figure 6. The relationship between ceramide levels and etoposide-induced cell death in LLC-PK1 cells. The cultured cells were grown to 70% confluency and deprived of FBS with the simultaneous addition of various concentrations of etoposide (10, 20, 50, 100 µM) for 24 hrs. The cell pellets were harvested, and the released-LDH assay (A) and ceramide analysis (B) were performed following the determination of total protein content. Values are expressed as the mean±
S.D. (n==3-5). Differences between untreated and treated samples with *P⁄0.05, **P⁄0.01 and **P⁄0.001 were defined as statistically significant.
100 µL of human plasma (data not shown). The spik- ed C17 ceramide standard in the samples enables the actual amount of endogenous ceramide to be measur- ed. This HPLC method is specific and more repro- ducible than previously published methods for the measurement of ceramide content. The introduction of a proper internal standard (C17 ceramide) into bio- logical samples improves the precision of the quanti- tative analysis of endogenously occurring C18 cera- mide and dihydroceramide. This greatly enhances the reproducibility of the results, which vary in other methods of ceramide quantification. One method for ceramide quantification employs E. coli DAG kinase
and [γ-32P] ATP to convert ceramide into [32P]cera- mide 1-phosphate7,14. However the 14-day half-life of
32P decreases the experimental flexibility and provid- es only a relative comparison of the radioactivity among samples.
In our method, possible contamination by sphin- goid bases in ceramide is excluded by TLC separa- tion. The efficient solvent system for TLC develop- ment allows the ceramide band to move far from the sphingosine and sphinganine bands. The Rfvalues for ceramide, sphingosine, and sphinganine are 0.54, 0.09, and 0.06, respectively (Figure 2). To verify that ceramide was not contaminated with sphingoid bases,
+FBS Vehicle ISP-1
-FBS
FBS Vehicle ISP-1
-FBS 0
1 2 3 4 5
Ceramide(nmol/mg protein)
(A)
(B)
Figure 7. Protection of LLC-PK1 cells from serum-deprivation-induced cell death by ISP-1 inhibition of ceramide biosynthesis. LLC-PK1 cells were grown to 50% confluency, deprived of FBS with the simultaneous addition of 1 µM ISP-1, a serine palmitoyltransferase inhibitor of the de novo sphingolipid biosynthetic pathway and incubated for 48 hrs.
Cell morphology was observed visually (A) and ceramide concentrations were determined by HPLC analysis (B). Values are expressed as the mean±S.D. (n==3-5). White arrowheads indicate the formation of characteristic dome in LLC-PK1 cells.
ceramide was measured following the extraction procedure with and without deacylation (data not shown). Contaminating sphingoid bases were not de- tected in any of the biological samples tested. Alth- ough OPA fluorescence detection is known to be specific for the amino group of analytes, the HPLC chromatogram shows many nonspecific peaks which interfere with the true sphingolipid peaks without the procedure of TLC purification (Figure 3A). Ceramide separated by TLC from crude lipid extract can be quantified without any interference of unknown peaks (Figure 3B), indicating that ceramides need to be purified for deacylation and HPLC analysis. Cera- mide deacylation by CDase occurred efficiently by fatty-acid-free BSA in enzymatic reaction buffer. The solubility of ceramide appeared to be enhanced by the complex with BSA, resulting in the increased amount of ceramides on the HPLC analysis (Figure 4). Ceramide analysis using more than 200µL of human plasma could not be performed in polypro- pylene 1.5-mL tubes. Large-scale analyses using glass tubes would be inconvenient and time-consuming, and might limit the number of samples. The increased sensitivity of our method allows small-scale analysis and can be applied to a large numbers of samples.
This analytical method is able to measure ceramides from a diversity of biological samples including cul- tured cells, mouse tissues, and mouse and human plasma (Table 2), and may be applicable to the use of ceramide as a biomarker for toxicity (Figure 6, 7) as well as for several human diseases19-22.
In this improved HPLC method, TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSA-complex leads to the increased deacylation. The use of an internal standard for the determination of ceramide concentration in samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples including cultured cells, mouse tissues, and mouse and human plasma.
Methods
Materials
ISP-1 (myriocin) and D-erythro-sphingosine were purchased from Biomol Research, Inc. (Plymouth Meeting, PA). N-oleoyl-D-erythro-sphingosine (C17
ceramide) was from Avanti Polar Lipids, Inc. (Ala- baster, AL). Pyridine and diisopropylether were from Sigma (St. Louis, MO). Fetal bovine serum (FBS) and culture medium for cell cultures were obtained from
Invitrogen (Gaithersburg, MD). HPLC-grade metha- nol was purchased from Merck (Darmstadt, Ger- many). o-Phthalaldehyde (OPA) was obtained from Molecular Probes, Inc. (Eugene, OR). Other reagents were of the highest purity available.
Cell Culture
Pig kidney epithelial (LLC-PK1) cells obtained from ATCC (Richmond, VA) were seeded at a density of 5×105 cells in flat-bottomed 6-well dishes and cultured in DMEM/F12 medium supplemented with 1.2 g/L sodium bicarbonate, 100 U/mL penicillin- streptomycin, and 5% (v/v) FBS at 37°C in a humidi- fied 5% CO2atmosphere. LLC-PK1 cells were grown to 50% confluence, deprived of FBS with the simul- taneous addition of 1 µM ISP-1, a serine palmitoy- ltransferase inhibitor of the de novo sphingolipid bio- synthetic pathway, and incubated for 48 hrs. Cells were harvested with a rubber policeman. Pellets of rat aortic vascular smooth muscle cells (VSMC) and HeLa cells were obtained from professor WY Park (Chungbuk National University, Korea).
Animals
The protocol was approved by the Ethical Com- mittee of the College of Pharmacy at Chungbuk National University. ICR mice (20 g body weight and 5-6 weeks-of-age) were obtained from Daehan Bio- link (Eumsung, Korea), and acclimated for 1 week in the animal facility (22°C, 12 hrs light/dark cycle).
Blood was collected and organs were removed for sphingolipid analysis.
Human Plasma
Blood samples were obtained from healthy volun- teers. Informed consent was obtained according to the Declaration of Helsinki before any blood was sampled. Plasma was prepared by centrifugation at 3,000×g for 10 min and stored at -20°C.
Lipid Extraction
Biological samples for lipid extraction were pellets of cultured cells and mouse tissues of 100 µg protein content and 50 µL of plasma from mice and humans.
Total lipid was extracted with 1 mL ethanol at 37°C for 1 hr following the addition of C17ceramide as the internal standard. The extract was centrifuged at 15,000×g for 10 min. The supernatant was dried in a Speed-Vac concentrator (Vision Scientific Co., Dae- jeon, Korea).
Thin Layer Chromatography (TLC)
The dry residue of the lipid extract was dissolved in 30 µL of chloroform/methanol (1 : 2, v/v) and spotted
on a high-performance thin-layer chromatography silica-gel plate (Merck, Darmstadt, Germany). The plate was developed in diisopropylether/methanol/
29% NH4OH (40 : 10 : 1, v/v/v). Ceramide standard lanes were cut from the sample lanes of the TLC plate and visualized by dipping the plate in 10% sul- furic acid and drying at 150°C. The areas in the sample lane with the same Rfvalues as the visualized band of C17ceramide standard were scraped off, and both ceramide and dihydroceramide were eluted with 1 mL methanol. The eluates were transferred to poly- propylene 1.5-mL tubes and dried in a Speed-Vac concentrator.
Enzymatic Deacylation
The ceramide residue was mixed with reaction buf- fer containing 25 mM Tris-HCl buffer, pH 7.5, 1%
sodium cholate, 15% fatty-acid-free BSA, and 150 µU CDase. Ceramide and dihydroceramide were deacy- lated into sphingosine and sphinganine, respectively, by CDase at 37°C for 6 hrs. BSA in the reaction buf- fer was precipitated by adding ethanol and removed by centrifugation, and the supernatant was dried.
HPLC Analysis
The sphingolipid extracts were dissolved in 120 µL methanol, mixed with 15 µL OPA reagent (50 mg OPA, 1 mL ethanol, 200 µL β-mercaptoethanol, and 50 mL 3% (w/v) boric acid buffer, pH 10.5), and incubated at room temperature for 30 min for deriva- tization. The HPLC analysis was performed using a Shimadzu (Tokyo, Japan) Model LC-10AT pump, a SIL-10AXL autosampler system, and an analytical Radial-Pak cartridge (Waters Associates, Inc., Milford, MA) packed with a Nova-Pak C18reversed-phase column (4 µm, 100 mm×8 mm). The isocratic mobile phase composition of methanol/distilled water/tri- ethylamine (92 : 8 : 0.1, v/v/v) and a flow rate of 1.0 mL/min were accurately controlled by the HPLC sys- tem controller (Shimadzu SCL-10A). The Shimadzu RF-10XLfluorescence detector was set at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The resulting data and chromatographic pro- files were evaluated using Borwin system manager software (JMBS, France).
Protein Assay
The total protein content of samples was determin- ed in order to normalize the results. The lysates from the cell pellets and tissues were solubilized with 0.2 N NaOH, mixed with PIERCE BCA reagents (Rock- ford, IL), and incubated for 30 min. The protein con- tent was quantified with a Molecular Devices ELISA reader (Sunnyvale, CA) at 562 nm based on the BSA
standard curve.
Statistics
All values were expressed as the means±standard deviations (SDs). Differences between untreated and treated samples were analyzed statistically by the unpaired Student’s t-test for single comparisons. Dif- ferences with *P⁄0.05, **P⁄0.01 and ***P⁄
0.001 were defined as statistically significant.
Acknowledgements
This work was supported by Korea Research Foun- dation Grant funded by the Korean Government (MOEHRD) (Basic Research Promotion Fund, KRF- 2004-E00250; The Regional Research Universities Program/Chungbuk BIT Research-Oriented Univer- sity Consortium), a grant (20070301-034-026) from BioGreen 21 Program, Rural Development Admini- stration, Republic of Korea and the research grant from Chungbuk National University in 2005.
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